Project summary Efficiency of CTL responses against viruses and cancer may vary and is often linked to MHC class I (MHC-I) variants and repertoire of available T-cell receptor (TCR). This suggests that parameters of TCR interactions with peptide-MHC (pMHC) ligands control the quality of CTL responses. Adoptive transfer of in vitro activated or engineered T cells with the specificity of interest has proved to be a useful modality to treat viral infection or cancer (1-3). However, this approach often causes off-target toxicity in vivo (4, 5). On the other hand, HIV elite controllers are capable of controlling the virus without any treatment (6, 7) and utilize selective clonotype of HIV-specific TCR genes (8). These and other findings led to a notion of an ?optimal? TCR (7). Similarly, optimal TCRs have been implicated in ?excellence? of cancer-specific T cells (10). Based on these and our previously published data (11-13), we hypothesized that a particular combination of TCR, peptide, and MHC-I variants determines the initiation of a rapid proximal signaling that is linked to efficient CTL response. To test the hypothesis, we will utilize HLA-A2 and HLA-B57 MHC-I proteins that contain mutations associated with either control of HIV infection (?favorable?) or those that are linked to HIV progression (?unfavorable?). We will solve crystal structures of selected cognate soluble scTCR bound to the pHLA-A2 or pHLA-B57 ligands that carry favorable or unfavorable mutations in the MHC moieties. Using X-ray data, we will delineate the mode of TCR engagement, i.e., relative TCR orientation over pMHC, and will calculate the pattern of molecular interactions at the interface of the TCRs with favorable and unfavorable pMHC proteins by means of Nobel Prize winning technique, Quantum mechanics/Molecular mechanics (QM/MM), using supercomputer available at Moscow University. We will then perform virtual TCR CDRs maturation to modify TCRs recognizing unfavorable pMHC and will select variants of the receptors whose interactions with unfavorable pMHC ligands would become favorable as established by QM/MM calculations, mode of TCR engagement, and measurements of the kinetics and the thermodynamics of the TCR-pMHC interactions. Results of the proposed experiments are expected (i) to reveal optimal mode of TCR engagement by pMHC that facilitates greater contribution of MHC-bound peptide to TCR binding and faster kinetics of TCR-pMHC interactions; (ii) to demonstrate the ability to convert unfavorable mode of TCR interactions to favorable one that is associated with an optimal TCR signaling and efficient CTL response. The novel strategy will allow to engineer effective virus- or cancer specific CTL that could be utilized for adoptive transfer therapy to treat viral infection and cancer.